Vaccine for prevention of necrotic enteritis in poultry

ABSTRACT

In certain embodiments, the present invention provides a poultry vaccine comprising an antigenic protein comprising a PlcC protein unit that is operably linked to a peptide linker that is operably linked to a NetB protein unit, where the vaccine is effective in stimulating a protective cellular and/or humoral immune response to  C. perfringens . Methods are also provided for making the vaccine and for vaccinating poultry by administering such a vaccine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/628,860, filed Jan. 6, 2020, which claims priority toInternational Application Number PCT/US2018/040632, filed Jul. 2, 2018,which claims the benefit of priority of U.S. Provisional ApplicationSer. No. 62/528,696, filed Jul. 5, 2017. The entire content of theapplications referenced above is hereby incorporated by referenceherein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 10, 2018 isnamed 17555_051WO1_SL.txt and is 45,056 bytes in size.

BACKGROUND

C. perfringens is a ubiquitous gram positive, spore-forming, anaerobicorganism, found in many environments surrounding poultry production,including soil, dust, feces, feed, litter, rodents, and the intestinalcontents of asymptomatic animals. C. perfringens is classified into fivegroups based on the types of toxins secreted, but only C. perfringenstype A strains are commonly associated with enteric disease in poultry.The toxins produced by type A C. perfringens strains cause necroticenteritis (NE) in colonized birds. Severe acute cases can result insudden death, while subclinical necrotic enteritis results in thickeningof the intestinal mucosa and decreased length of microvilli in theileum. The collective impact of C. perfringens colonization is to reducethe absorptive surface in the intestinal tract with a consequentreduction in the ability of birds to benefit from nutrients in food,resulting in a reduced rate of growth. C. perfringens also inducescellulitis and gangrenous dermatitis and is becoming an increasingconcern in turkeys as well.

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 62/528,696, filed Jul. 5, 2017, which applicationis incorporated by reference herein.

C. perfringens infections and NE have been traditionally controlled byaddition of Antimicrobial Growth Promoters (AGP) and coccidiostats inthe animal feed. Large quantities of antimicrobials were used as AGP andas prophylaxis against enteric bacterial pathogens, including C.perfringens. The use of AGP has been condemned due to concerns aboutincreased antibiotic resistance in human pathogens. Consequently, anincrease in the incidence of sub-clinical NE is linked to the withdrawalof AGP. This had been observed initially in Scandinavian countriesfollowing the ban on AGP in the early 1990s. Furthermore, the decline inuse of ionophore coccidiostats, which can prevent C. perfringenslesions, has exacerbated the resurgence of NE. Recent moves by the UScongress and the FDA to restrict the use of growth promoting antibioticsand public pledges by major poultry consumers (e.g. McDonald's, Costco,Chick-fil-A) to eliminate antibiotic-fed poultry from their menus,indicates that the old practices are on the way out. Thus, NE is are-emerging disease and a threat to the current objective of“antimicrobial-free” poultry farming.

Most birds infected with virulent strains remain asymptomatic and showreduced growth performance. C. perfringens can cause a range of healthproblems in infected birds, ranging from a subclinical infection whichcan result in poor feed conversion caused by decreased digestion andadsorption, to necrotic enteritis, resulting in a variety of symptomsincluding severe depression, decreased appetite, reluctance to move,diarrhea and ruffled feathers, often leading to death. Clinical illnessis usually short, with birds often simply found dead. Onset of diseasesymptoms generally occurs in broilers from two to five weeks of age,coinciding with the disappearance of maternal antibodies. However, NEhas also been reported in layers of various ages. Gross lesionstypically involve the duodenum, jejunum and sometimes the ileum,although even cecal lesions can occur. Intestines are friable anddistended with gas and fluid and a diphtheritic membrane is often foundin the mucosa. Subclinical infection with C. perfringens can lead toeconomic losses, due to reduced growth rates and poor feed conversion.It is likely that losses due to subclinical infections may constitute alarger problem overall than losses due to acute disease. Occasionally,cholangiohepatitis can result, leading to condemnation losses.

Diet has also been implicated as a factor that can predispose birds toNE. Inclusion of wheat, rye, barley, oat groats or fish meal in the dietcan lead to increased numbers of C. perfringens and incidence of NE.Dietary fat source can also influence the C. perfringens population(19). Current thinking is that predisposing factors such as a highprotein diet (e.g. fishmeal) and/or Eimeria infection result inalterations of the chicken gut microbiota that allow incoming pathogenicC. perfringens strains to become established.

Thus, there is an unmet need for an improved, effective vaccine againstC. perfringens that protects the birds against the disease. A vaccinethat would protective immunity would meet this need.

SUMMARY

Necrotic enteritis caused by Clostridium perfringens is a seriouseconomic problem in the broiler industry, with losses up to $2 billionannually. In the US, this problem is likely to be exacerbated as the useof antibiotics in poultry rearing is phased out. This inventiondescribes a method to produce in plants, a novel protein fusing twotoxoids that elicit immune responses protective against necroticenteritis. The fusion protein antigen can be purified from plant cellsfor use as an injectable vaccine or can by directly applied to poultryfeed for use as an oral vaccine.

In certain embodiments, the present invention provides an antigenicprotein comprising a PlcC protein unit that is operably linked to apeptide linker that is operably linked to a NetB protein unit.

In certain embodiments, the present invention provides a nucleic acidencoding the antigenic protein described herein.

In certain embodiments, the present invention provides an expressioncassette comprising the nucleic acid described herein and a promoter.

In certain embodiments, the present invention provides a recombinantvector comprising the expression cassette described herein and a vector.

In certain embodiments, the present invention provides a plant cellcomprising the antigenic protein described herein, the nucleic aciddescribed herein, the expression cassette described herein or therecombinant vector described herein.

In certain embodiments, the present invention provides animal feedcomprising the plant cell described herein.

In certain embodiments, the present invention provides a vaccinecomprising the antigenic protein described herein, the nucleic aciddescribed herein, the expression cassette described herein, therecombinant vector described herein, the plant cell described herein, orthe animal feed described herein.

In certain embodiments, the present invention provides a method ofprotecting an avian species from C. perfringens infections comprisingadministering the vaccine described herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Fibrin deposits and lesions on the intestinal tract of a chickenwith necrotic enteritis.

FIG. 2. Schematic of PlcC-NetB fusion protein.

FIG. 3. Western blot of plant-made PlcC-NetB fusion and NetB proteins.Extracts of N. benthamiana leaf samples expressing either PlcC-NetBfusion or NetB proteins were resolved by SDS-PAGE and electro-blotted toa PVDF membrane and probed with rabbit anti-NetB serum. For NetB, 4different forms can be observed, with the smallest at ˜34 kDa (thetheoretical size of unglycosylated NetB) and the largest ˜46 kDa,suggesting glycosylation at all 4 potential Asn-linked sites. ForPlcC-NetB fusion protein, the theoretical size of unglycosylated proteinis ˜49 kDa; the observed bands occur between ˜52 kDa and 62 kDa, withthe largest suggesting glycosylation at all 5 potential Asn-linkedsites.

FIGS. 4A-4C. IgY serum titers from immunized and non-immunized birds inExperiment 1. A paired t-test was performed between chickens thatreceived the fusion protein and non-vaccinated control group. FIG. 4A.PlcC0NetB titers; FIG. 4B. PlcC titers; FIG. 4C. NetB titers. * P≤0.009.

FIG. 5. Summary of lesion scores from Experiments 1 and 2.

FIG. 6. Map of pBYR2eK2M-6HplcCnetB.

DETAILED DESCRIPTION

Necrotic enteritis (NE) is caused by type A strains of the bacteriumClostridium perfringens. The total global economic losses to the poultryindustry due to NE is estimated to be over 2 billion dollars annually.C. perfringens produces two toxins, alpha-toxin and NetB. The NetB toxinis responsible for the symptoms associated with NE and anti-NetBantibodies are protective. Immune responses against alpha-toxin arepartially protective despite the fact that it does not play a directrole in NE. We describe a single fusion protein combining immunogenicand non-toxic components of alpha-toxin and NetB that can be used toimmunize poultry against NE. The fusion protein is produced in plantswhich can be purified and used as an injectable preparation or feddirectly to poultry to elicit a protective immune response.

The use of plants for the production and oral delivery of a necroticenteritis vaccine is novel. Moreover, the PlcC-NetB fusion proteindescribed here is novel and is strongly expressed in plants, which lackcholesterol and thus may be immune to the toxic effects of NetB, thuspermitting the observed high expression level.

This novel fusion protein combines the two most potent protectiveantigens against necrotic enteritis, NetB and alpha-toxin, into a singleantigenic protein. NetB is the toxin responsible for necrotic enteritissymptoms. The alpha-toxin (Plc) may also contribute to disease, and, inaddition, antibodies against alpha-toxin are targeted to the surface ofC. perfringens, inhibiting its growth. However, strains lackingalpha-toxin can cause disease, such that a vaccine relying only onimmune responses against alpha-toxin will not provide protection againstthese strains. Combining both alpha-toxin and NetB epitopes will providerobust protection against disease. The PlcC-NetB protein can be purifiedfrom plants, in either a glycosylated or non-glycosylated form, and usedas an injectable vaccine to protect birds directly. Injection into henswill protect their offspring in the first two weeks of life via maternalantibodies passed on in the egg. When this protein is produced in a foodplant, such as corn, with or without an LT adjuvant, the resultingrecombinant plant can be applied directly to the feed, resulting in apotentially low cost, oral vaccine to protect chickens against necroticenteritis.

The use of plants for the production and oral delivery of a necroticenteritis vaccine is novel. Moreover, the PlcC-NetB fusion proteindescribed here is novel and is strongly expressed in plants, which lackcholesterol and thus may be immune to the toxic effects of NetB, thuspermitting the observed high expression level.

C. perfringens type A strains produce alpha-toxin, a membrane-damagingphospholipase C enzyme. The toxin is hemolytic, necrotizing and lethal.It is the toxin that responsible for C. perfringens-mediated gasgangrene. Many of the symptoms of NE can be reproduced with culture-freesupernatants of C. perfringens. Since these supernatants were known tocontain alpha-toxin, it was assumed that alpha-toxin was responsible.More recent studies have identified a novel toxin linked to necroticenteritis, designated NetB toxin. It was first identified in a virulentC. perfringens type A strain isolated in Australia and it has beendetected in the vast majority of NE-associated C. perfringens strainsthroughout the world. Thus, it is now considered to be the most criticalvirulence factor for the development of NE in broilers. NetB is apore-forming toxin encoded on a large conjugative plasmid (approximately85 kb) within a 42 kilobase (kb) pathogenicity locus (NELoc-1), showingsimilarity to C. perfringens β-toxin (38% identity). The presence ofnetB gene is highly correlated with necrotic enteritis strains. NetB isalso a protective antigen, particularly in combination with otherimmunogenic components. One study showed that the levels of serumantibodies against both alpha-toxin and NetB toxin were significantlyhigher in apparently healthy chickens compared to birds with clinicalsigns of NE, suggesting that these antitoxin antibodies play a role inprotection. The large clostridial cytotoxin TpeL (predicted molecularmass=191 kDa), first identified in type C strains, is also produced bysome type A strains and has been linked to increased virulence,particularly in strains producing netB. However, it should be noted thata recent study of historical NE strains collected >15 years ago inAlabama revealed a low prevalence of the netB gene, indicating that netBmay be dispensable for some NE for some strains or in some situations.Nevertheless, it is clear that the overwhelming majority of currentnecrotic enteritis strains produce this toxin.

Toxins have traditionally been targeted as antigens of interest forcontrolling clostridial infections. The C. perfringens alpha-toxin (Plc)is the major virulence determinant for gas gangrene and antibodies to C.perfringens alpha-toxin prevent gas gangrene in mice. The C. perfringensgene encoding alpha-toxin is plc (for phospholipase C). The protein isdivided into two domains, the amino-terminal domain encodes thecatalytic site responsible for phospholipase activity, while thecarboxy-terminal domain is involved in interactions with phospholipids,targeting the enzyme to host cell membranes. The alpha-toxincarboxy-terminal fragment (amino acids 247-370) is non-toxic andimmunization with this fragment confers protection against alpha-toxinand C. perfringens in a gas gangrene mouse model. Immune responsesagainst the C-terminal domain, PlcC, can provide protection againstsubsequent challenge with C. perfringens.

NetB binds to cholesterol in membranes, forming heptameric pores. Anumber of single amino acid substitutions in the rim loop region cansignificantly reduce its ability to bind to cells and its toxicity.These include Y191A, R200A, W257A and W262A, S254L, R230Q and W287R.Some of these were shown to retain the ability to generate protectiveimmune responses, including W262A and S254L. A number of studies havedemonstrated the potential of vaccination to control NE. A vaccineutilizing detoxified alpha-toxin can induce some protection againstexperimental infection. Since alpha-toxin is not required in order forC. perfringens to cause NE in chickens, it is not clear whyalpha-toxoids are protective. One likely explanation is based on datashowing that anti-alpha-toxin (anti-Plc) antibodies bind to the surfaceof Plc+C. perfringens strains and that these antibodies can also inhibitC. perfringens growth. Thus, it is possible that the reason anti-Plcantibodies are protective is due to their growth inhibitory propertiesand not directly due to detoxification.

NetB is also a protective antigen, which could provide significantprotection against NE challenge, especially in combination with otherimmunogenic components. Both alpha-toxin (C-fragment) and NetB (W262A)toxoids were combined (30 g of each) in Quil A adjuvant and used tosubcutaneously inject broiler birds 3 times, on days 3, 9 and 15. Birdsinjected with only one of the proteins were also included. The immunizedbirds were partially protected against a mild challenge (gavage only),but not against a more severe, in feed challenge. In some studies, henswere infected with NetB toxoid and antibodies against NetB weretransferred from immunized hens to their progeny, providing protectionto the chicks against C. perfringens challenge. In another study,immunization with both NetB and alpha-toxin toxoids using a liveSalmonella delivery vector induced mucosal antibodies against bothtoxins and elicited a protective response. S. Typhimurium vaccine trainsengineered to deliver both toxoids provided significantly betterprotection than strains delivering each toxin alone.

Recently an injectable alpha-toxoid preparation produced by Intervet,called Netvax, has come on the market for use in broiler breeders toincrease protection in chicks during the first few weeks of life.However, there is no commercial vaccine that includes a NetB immunogeniccomponent. Several vaccine antigens have been stably expressed in cornand rice, which are convenient for use in feed products. Despiteconcerns regarding oral tolerance, feeding animals plant-based vaccineshas been shown to be effective in agricultural animals, includingpoultry.

Proteins

In certain embodiments, the present invention provides an antigenicprotein comprising a PlcC protein unit that is operably linked to apeptide linker that is operably linked to a NetB protein unit.

In certain embodiments, the PlcC protein unit, the peptide linker andthe NetB protein unit each have an N-terminus and a C-terminus, andwherein the C-terminus of the PlcC protein unit is linked to theN-terminus of the peptide linker, and the C-terminus of the peptidelinker is operably linked to the N-terminus of the NetB protein unit.

In certain embodiments, the PlcC protein unit has at least 95% sequenceidentity to SEQ ID NO: 3. The PlcC protein unit of SEQ ID NO: 3 is aminoacids 248-370 of alpha toxin (GenBank accession AAP-15462.1).

In certain embodiments, the PlcC protein unit has 100% sequence identityto SEQ ID NO: 3.

In certain embodiments, the NetB protein unit has at least 95% sequenceidentity to SEQ ID NO: 5. The NetB protein unit of SEQ ID NO: 5 is aminoacids 31-322 of (GenBank accession ACN73257.1).

In certain embodiments, the NetB protein unit has one or more amino acidsubstitutions at Y191A, R200A, W257A and W262A, S254L, R230Q or W287R ofSEQ ID NO: 5.

In certain embodiments, the NetB protein unit has 100% sequence identityto SEQ ID NO: 5.

In certain embodiments, the peptide linker has at least 95% sequenceidentity to SEQ ID NO: 4.

In certain embodiments, the peptide linker has 100% sequence identity toSEQ ID NO: 4.

In certain embodiments, the antigenic protein further comprises a 6Histtag having an N-terminus and a C-terminus, wherein the C-terminus of the6Hist tag is operably linked to the N-terminus of the PlcC protein unit.

In certain embodiments, the 6His tag has 100% identity to SEQ ID NO: 2.

In certain embodiments, the antigenic protein further comprises a plantsignal peptide having an N-terminus and a C-terminus, wherein theC-terminus of the plant signal peptide is operably linked to theN-terminus of the 6Hist tag.

In certain embodiments, the plant signal peptide has at least 95%sequence identity to SEQ ID NO: 1.

In certain embodiments, the plant signal peptide has 100% sequenceidentity to SEQ ID NO: 1.

The term “amino acid” includes the residues of the natural amino acids(e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as wellas unnatural amino acids (e.g., phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). Theterm also includes peptides with reduced peptide bonds, which willprevent proteolytic degradation of the peptide. Also, the term includesthe amino acid analog α-amino-isobutyric acid. The term also includesnatural and unnatural amino acids bearing a conventional aminoprotecting group (e.g., acetyl or benzyloxycarbonyl), as well as naturaland unnatural amino acids protected at the carboxy terminus (e.g., as a(C₁-C₆)alkyl, phenyl or benzyl ester or amide; or as an α-methylbenzylamide). Other suitable amino and carboxy protecting groups are known tothose skilled in the art (See for example, T. W. Greene, ProtectingGroups In Organic Synthesis; Wiley: New York, 1981, and references citedtherein).

A “variant” of one of the proteins that one that is not completelyidentical to a native protein. Such variant protein can be obtained byaltering the amino acid sequence by insertion, deletion or substitutionof one or more amino acid. The amino acid sequence of the protein ismodified, for example by substitution, to create a polypeptide havingsubstantially the same or improved qualities as compared to the nativepolypeptide. The substitution may be a conserved substitution. A“conserved substitution” is a substitution of an amino acid with anotheramino acid having a similar side chain. A conserved substitution wouldbe a substitution with an amino acid that makes the smallest changepossible in the charge of the amino acid or size of the side chain ofthe amino acid (alternatively, in the size, charge or kind of chemicalgroup within the side chain) such that the overall peptide retains itsspacial conformation but has altered biological activity. For example,common conserved changes might be Asp to Glu, Asn or Gln; His to Lys,Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Aline iscommonly used to substitute for other amino acids. The 20 essentialamino acids can be grouped as follows: alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan and methionine havingnonpolar side chains; glycine, serine, threonine, cystine, tyrosine,asparagine and glutamine having uncharged polar side chains; aspartateand glutamate having acidic side chains; and lysine, arginine, andhistidine having basic side chains.

The amino acid changes are achieved by changing the codons of thecorresponding nucleic acid sequence. It is known that such polypeptidescan be obtained based on substituting certain amino acids for otheramino acids in the polypeptide structure in order to modify or improvebiological activity. For example, through substitution of alternativeamino acids, small conformational changes may be conferred upon apolypeptide that results in increased activity. Alternatively, aminoacid substitutions in certain polypeptides may be used to provideresidues, which may then be linked to other molecules to providepeptide-molecule conjugates which retain sufficient properties of thestarting polypeptide to be useful for other purposes.

One can use the hydropathic index of amino acids in conferringinteractive biological function on a polypeptide, wherein it is foundthat certain amino acids may be substituted for other amino acids havingsimilar hydropathic indices and still retain a similar biologicalactivity. Alternatively, substitution of like amino acids may be made onthe basis of hydrophilicity, particularly where the biological functiondesired in the polypeptide to be generated is intended for use inimmunological embodiments. The greatest local average hydrophilicity ofa “protein”, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity. Accordingly, it is noted thatsubstitutions can be made based on the hydrophilicity assigned to eachamino acid.

In using either the hydrophilicity index or hydropathic index, whichassigns values to each amino acid, it is preferred to conductsubstitutions of amino acids where these values are ±2, with ±1 beingparticularly preferred, and those with in ±0.5 being the most preferredsubstitutions.

The variant protein has at least 80%, at least about 90%, or even atleast about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than100%, contiguous amino acid sequence homology or identity to the aminoacid sequence of a corresponding native protein.

A variant may include amino acid residues not present in thecorresponding native protein or deletions relative to the correspondingnative protein. A variant may also be a truncated “fragment” as comparedto the corresponding native protein, i.e., only a portion of afull-length protein. Protein variants also include peptides having atleast one D-amino acid.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

Amino acid sequence of 6H-plcC-netB fusion protein:

(SEQ ID NO: 13) mankhlslslflyllglsaslasgHHHHHHgsDPSVGNNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMAGSKDTYTFKLKDENLKIDDIQNMWIRKRKYTAFPDAYKPENIKVIANGKVVVDKDINEWISGNSTYNIKggsggsggpsggsggsELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETTQARGTNKLSSTSEYNEFMFK INWQDHKIEYYL

Coding of Regions:

Lower case, regular type=ER signal peptide from barley alpha amylasegene

Upper case, Italics=6-His metal affinity tag

Lower case, italics=linker sequences

Upper case, regular type=plcC

Upper case, Bold type=netB (W262A mutation underlined)

In certain embodiments, e.g., for cytosolic instead of ER targeting, theN-terminal signal peptide (Lower case, regular type) is omitted.

Nucleic Acid

In certain embodiments, the present invention provides a nucleic acidencoding the antigenic protein described herein.

In certain embodiments, the nucleic acid has been plant-codon optimizedfor plant expression.

In certain embodiments, the nucleic acid has been plant-codon optimizedfor expression in Nicotiana benthamiana or Arabidopsis.

In certain embodiments, the nucleic acid has at least 95% sequenceidentity to SEQ ID NO: 6.

In certain embodiments, the nucleic acid has 100% sequence identity toSEQ ID NO: 6.

The fusion protein gene for the 6H-plcC-netB fusion protein (SEQ ID NO:13 (amino acid sequence)) was codon optimized for expression inNicotiana benthamiana. Each codon was assessed for its preference of usein highly expressed genes of N. benthamiana, N. tabacum, and Arabidopsisthaliana, using coding sequences obtained from Genbank accessions(Geyer, B. C., Kannan, L., Cherni, I., Woods, R. R., Soreq, H., and Mor,T. S. (2010) Transgenic plants as a source for the bioscavenging enzyme,human butyrylcholinesterase. Plant Biotechnol J. 8(8):873-86). Use of aparticular codon was avoided if it represented less than 50% of thefrequency of the most preferred synonymous codon in the reference geneset. The remaining codons were used at frequencies proportional to theirfrequencies of use in the reference gene set. Furthermore, A-richsequences that could function as a polyadenylation near-upstream elementwere avoided, including AATAAA, AATGGA, AATGAA, TATAAA, AATAAT, AATAAG,AATATT, GATAAA, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT,ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, CATAAA, ACTAAA, andAAAAAA. Sequences that could function as 5′ intron splice recognitionsequences were avoided, including GTAACA, GTGCTC, GTTAGT, GTAAAT,GTAAAG, GTCTGT, GTAAGG, GTGAGT, GTAAAA, GTAAGT, GTAAGC, GTACGT, GTAACT,GTAAGA, GTTAAA, GTAATA, and GTACAT. As much as possible, sequences thatcould function as 3′ intron splice recognition sequences were avoided,including GCAGG, CCAGG, TCAGG, ATAGG, GTAGG, TTAGG, ACAGG, and AAAGG.Potential RNA destabilizing sequences were avoided, including ATTTA,TAGATY, ATAGAT, and TTTTTT. Potential termination signals for RNApolymerase II were avoided as much as possible, includingCA(N7-9)AGTNNA. The potential DNA C-methylation signal CCGG was avoidedas much as possible.

The nucleotide sequence of pBYR2eK2M-6HplcCnetB is the following (FIG. 6):(SEQ ID NO: 14)CGATCGGTCGATTCATAGAAGATTAGATTTTTCATAGTATTTTTTTAAAGTAAACCTTTAACTACGGTTAGGACACTTTTAAGTTAAATTTAATTTGAACCCTTAAATTAATTTTTAAAATAGATAAATATCAATCATCCTGATATGCTTTTGAAAAAATGAATGAGAAAGATGATTCAATTAAGGCCACATTTTAATCATGACTAAAATAATATACAGTATAATTTCATATATATTTGCTTTAAAAAAAAATTGACAATCCATTCGTTTCTAGCAATAAATTTCTTCAACCACAAATATATTAAAGATAACTACGGCATAGAAACAAAAATCTATGAAGAATTTTTGTATACTTCATATGAAATTAAAAAAAACTTCATTGAACATCAAAATAATAATAATAATCATAAACTCCTCAATATTTATATTCCTAGCTTCTTGAATTAAATTGTTTACATATTCAACGATGTAAAAAATTATTTCTCTATCTATTTTCCTTATATCATGCATGGTTTCACATATATCAAAGGATAAAAGCAATCTATGTAAATTATCTCACTTTATTAAGTTTTCTATCTGAATTATTGAGAACGTAGATTTCTTTTTGCACTATCCCCCAATAATTAGCAAAACACACCTAGACTAGATTTGTTTTGCTAACCCAATTGATATTAATTATATATGATTAATATTTATATGTATATGGAATTGGTTAATAAAATGCATCTGGTTCATCAAAGAATTATAAAGACACGTGACATTCATTTAGGATAAGAAATATGGATGATCTCTTTCTCTTATTCAGATAATTAGTAATTACACATAACACACAACTTTGATGCCCACATTATAGTGATTAGCATGTCACTATGTGTGCATCCTTTTATTTCATACATTAATTAACTTGGCCAATCCAGAAGATGGACAAGTCTAGGGTCACATTGCAGGGTACTCTAGCTTACTCGCCTTCTTTTTCGAAGGTTTGAGTACCTTCAGGGCATCCTCTTGATACATTACTTTCCACTTCGATTGGGGCAAGCTGTAGCAGTTCTTGCTTAGACCGAATTGCCATCTCACAGAGATGCTGAAGAGTTCGCGACCCTCCAGAAACGGTGATACTAACTCCTCGAAACCGAATACTATAGGTACATCCGATCTGGTCGAAACCGAAAAATCGAGATGCTGCATAGTTAACCGAATCTCCCGTCCAAGATCCAAGGACTCTGTGCAGTGAAGCTTCCGTCCTGTCGTATCTGAGATATCTCTTAAATACAACTTTCCCGAAACCCCAGCTTTCCTTGAAACCAAGGGGATTATCTTGATTCGAATTCGTCTCATCGTTATGTAGCCGCCACTCAGTCCAACTCGGACTTTCGTCAGGAAGTTTGAAGGGAGAAGTTGTACCTCCTGATCCTCCATCCCAACGTTCACTGTTAGCTTGTTCCCTAGCGTCGTTTCCTTGTATAGCTCGTTCCATGGATTGTAAATAGTAATTGTAATGTTGTTTGTTGTTTGTTGTTGTTGGTAATTGTTGTAAAAATACGCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTCAACGATGGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGAGCCACCTTCCTTTTCCACTATCTTCACAATAAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCGGATATTACCCTTTGTTGAAAAGTCTCAATTGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTTTTGGAGTAGACAAGTGTGTCGTGCTCCACCATGTTCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGTTCCGCTTCCTTTAGCAGCCCTTGCGCCCTGAGTGCTTGCGGCAGCGTGAAGCTGGCGCGCCGCTCTAGCAGAAGGCATGTTGTTGTGACTCCGAGGGGTTGCCTCAAACTCTATCTTATAACCGGCGTGGAGGCATGGAGGCAAGGGCATTTTGGTAATTTAAGTAGTTAGTGGAAAATGACGTCATTTACTTAAAGACGAAGTCTTGCGACAAGGGGGGCCCACGCCGAATTTTAATATTACCGGCGTGGCCCCACCTTATCGCGAGTGCTTTAGCACGAGCGGTCCAGATTTAAAGTAGAAAAGTTCCCGCCCACTAGGGTTAAAGGTGTTCACACTATAAAAGCATATACGATGTGATGGTATTTGATAAAGCGTATATTGTATCAGGTATTTCCGTCGGATACGAATTATTCGTACGACCCTCCTGCAGGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACCTCGAGAAACAAACAAAATCAACAAATATAGAAAATAACGCATTTCCAATTCTTTGAAATTTCTGCAACATCTAGAACAATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAAGGAGGTTCTGGTGGATCAGGAGGTCCATCTGGAGGTTCTGGAGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTTTAAGAGCTCGAAGTGACATCACAAAGTTGAAGGTAATAAAGCCAAATTAATTAAGACATTTTCATAATGATGTCAAGAATGCAAAGCAAATTGCATAACTGCCTTTATGCAAAACATTAATATAATATAAATTATAAAGAACTGCGCTCTCTGCTTCTTATTTTCTTAGCTTCATTTATTAGTCACTAGCTGTTCAGAATTTTCAGTATCTTTTGATATTACTAAGAACCTAATCACACAATGTATATTCTTATGCAGGAAAAGCAGAATGCTGAGCTAAAAGAAAGGCTTTTTCCATTTTCGAGAGACAATGAGAAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAAAGAGTAAATAATAAAGCCCCACAGGAGGCGAAGTTCTTGTAGCTCCATGTTATCTAAGTTATTGATATTGTTTGCCCTATATTTTATTTCTGTCATTGTGTATGTTTTGTTCAGTTTCGATCTCCTTGCAAAATGCAGAGATTATGAGATGAATAAACTAAGTTATATTATTATACGTGTTAATATTCTCCTCCTCTCTCTAGCTAGCCTTTTGTTTTCTCTTTTTCTTATTTGATTTTCTTTAAATCAATCCATTTTAGGAGAGGGCCAGGGAGTGATCCAGCAAAACATGAAGATTAGAAGAAACTTCCCTCTTTTTTTTCCTGAAAACAATTTAACGTCGAGATTTATCTCTTTTTGTAATGGAATCATTTCTACAGTTATGACGAATTCTCGATTAAAAATCCCAATTATATTTGGTCTAATTTAGTTTGGTATTGAGTAAAACAAATTCGAACCAAACCAAAATATAAATATATAGTTTTTATATATATGCCTTTAAGACTTTTTATAGAATTTTCTTTAAAAAATATCTAGAAATATTTGCGACTCTTCTGGCATGTAATATTTCGTTAAATATGAAGTGCTCCATTTTTATTAACTTTAAATAATTGGTTGTACGATCACTTTCTTATCAAGTGTTACTAAAATGCGTCAATCTCTTTGTTCTTCCATATTCATATGTCAAAATCTATCAAAATTCTTATATATCTTTTTCGAATTTGAAGTGAAATTTCGATAATTTAAAATTAAATAGAACATATCATTATTTAGGTATCATATTGATTTTTATACTTAATTACTAAATTTGGTTAACTTTGAAAGTGTACATCAACGAAAAATTAGTCAAACGACTAAAATAAATAAATATCATGTGTTATTAAGAAAATTCTCCTATAAGAATATTTTAATAGATCATATGTTTGTAAAAAAAATTAATTTTTACTAACACATATATTTACTTATCAAAAATTTGACAAAGTAAGATTAAAATAATATTCATCTAACAAAAAAAAAACCAGAAAATGCTGAAAACCCGGCAAAACCGAACCAATCCAAACCGATATAGTTGGTTTGGTTTGATTTTGATATAAACCGAACCAACTCGGTCCATTTGCACCCCTAATCATAATAGCTTTAATATTTCAAGATATTATTAAGTTAACGTTGTCAATATCCTGGAAATTTTGCAAAATGAATCAAGCCTATATGGCTGTAATATGAATTTAAAAGCAGCTCGATGTGGTGGTAATATGTAATTTACTTGATTCTAAAAAAATATCCCAAGTATTAATAATTTCTGCTAGGAAGAAGGTTAGCTACGATTTACAGCAAAGCCAGAATACAAAGAACCATAAAGTGATTGAAGCTCGAAATATACGAAGGAACAAATATTTTTAAAAAAATACGCAATGACTTGGAACAAAAGAAAGTGATATATTTTTTGTTCTTAAACAAGCATCCCCTCTAAAGAATGGCAGTTTTCCTTTGCATGTAACTATTATGCTCCCTTCGTTACAAAAATTTTGGACTACTATTGGGAACTTCTTCTGAAAATAGTGGTACCGAGTGTACTTCAAGTCAGTTGGAAATCAATAAAATGATTATTTTATGAATATATTTCATTGTGCAAGTAGATAGAAATTACATATGTTACATAACACACGAAATAAACAAAAAAACACAATCCAAAACAAACACCCCAAACAAAATAACACTATATATATCCTCGTATGAGGAGAGGCACGTTCAGTGACTCGACGATTCCCGAGCAAAAAAAGTCTCCCCGTCACACATATAGTGGGTGACGCAATTATCTTCAAAGTAATCCTTCTGTTGACTTGTCATTGATAACATCCAGTCTTCGTCAGGATTGCAAAGAATTATAGAAGGGATCCCACCTTTTATTTTCTTCTTTTTTCCATATTTAGGGTTGACAGTGAAATCAGACTGGCAACCTATTAATTGCTTCCACAATGGGACGAACTTGAAGGGGATGTCGTCGATGATATTATAGGTGGCGTGTTCATCGTAGTTGGTGAAGTCGATGGTCCCGTTCCAGTAGTTGTGTCGCCCGAGACTTCTAGCCCAGGTGGTCTTTCCGGTACGAGTTGGTCCGCAGATGTAGAGGCTGGGGTGTCTGACCCCAGTCCTTCCCTCATCCTGGTTAGATCGGCCATCCACTCAAGGTCAGATTGTGCTTGATCGTAGGAGACAGGATGTATGAAAGTGTAGGCATCGATGCTTACATGATATAGGTGCGTCTCTCTCCAGTTGTGCAGATCTTCGTGGCAGCGGAGATCTGATTCTGTGAAGGGCGACACGTACTGCTCAGGTTGTGGAGGAAATAATTTGTTGGCTGAATATTCCAGCCATTGAAGCTTTGTTGCCCATTCATGAGGGAACTCTTCTTTGATCATGTCAAGATACTCCTCCTTAGACGTTGCAGTCTGGATAATAGTTCGCCATCGTGCGTCAGATTTGCGAGGAGACACCTTATGATCTCGGAAATCTCCTCTGGTTTTAATATCTCCGTCCTTTGATATGTAATCAAGGACTTGTTTAGAGTTTCTAGCTGGCTGGATATTAGGGTGATTTCCTTCAAAATCGAAAAAAGAAGGATCCCTAATACAAGGTTTTTTATCAAGCTGGATAAGAGCATGATAGTGGGTAGTGCCATCTTGATGAAGCTCAGAAGCAACACCAAGGAAGAAAATAAGAAAAGGTGTGAGTTTCTCCCAGAGAAACTGGAATAAATCATCTCTTTGAGATGAGCACTTGGGGTAGGTAAGGAAAACATATTTAGATTGGAGTCTGAAGTTCTTGCTAGCAGAAGGCATGTTGTTGTGACTCCGAGGGGTTGCCTCAAACTCTATCTTATAACCGGCGTGGAGGCATGGAGGCAAGGGCATTTTGGTAATTTAAGTAGTTAGTGGAAAATGACGTCATTTACTTAAAGACGAAGTCTTGCGACAAGGGGGGCCCACGCCGAATTTTAATATTACCGGCGTGGCCCCACCTTATCGCGAGTGCTTTAGCACGAGCGGTCCAGATTTAAAGTAGAAAAGTTCCCGCCCACTAGGGTTAAAGGTGTTCACACTATAAAAGCATATACGATGTGATGGTATTTGATGGAGCGTATATTGTATCAGGTATTTCCGTCGGATACGAATTATTCGTACGGCCGGACCGGTCCCCTAGGCCGGCCAATTCGAGATCGGCCGCGGCTGAGTGGCTCCTTCAATCGTTGCGGTTCTGTCAGTTCCAAACGTAAAACGGCTTGTCCCGCGTCATCGGCGGGGGTCATAACGTGACTCCCTTAATTCTCCGCTCATGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAATCGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCAGATCTGGCGCCGGCCAGCGAGACGAGCAAGATTGGCCGCCGCCCGAAACGATCCGACAGCGCGCCCAGCACAGGTGCGCAGGCAAATTGCACCAACGCATACAGCGCCAGCAGAATGCCATAGTGGGCGGTGACGTCGTTCGAGTGAACCAGATCGCGCAGGAGGCCCGGCAGCACCGGCATAATCAGGCCGATGCCGACAGCGTCGAGCGCGACAGTGCTCAGAATTACGATCAGGGGTATGTTGGGTTTCACGTCTGGCCTCCGGAGACTGTCATACGCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGCAGTTGCCATGTTTTACGGCAGTGAGAGCAGAGATAGCGCTGATGTCCGGCGGTGCTTTTGCCGTTACGCACCACCCCGTCAGTAGCTGAACAGGAGGGACAGCTGATAGACACAGAAGCCACTGGAGCACCTCAAAAACACCATCATACACTAAATCAGTAAGTTGGCAGCATCACCCATAATTGTGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTTAAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGATGTGGCGCAACGATGCCGGCGACAAGCAGGAGCGCACCGACTTCTTCCGCATCAAGTGTTTTGGCTCTCAGGCCGAGGCCCACGGCAAGTATTTGGGCAAGGGGTCGCTGGTATTCGTGCAGGGCAAGATTCGGAATACCAAGTACGAGAAGGACGGCCAGACGGTCTACGGGACCGACTTCATTGCCGATAAGGTGGATTATCTGGACACCAAGGCACCAGGCGGGTCAAATCAGGAATAAGGGCACATTGCCCCGGCGTGAGTCGGGGCAATCCCGCAAGGAGGGTGAATGAATCGGACGTTTGACCGGAAGGCATACAGGCAAGAACTGATCGACGCGGGGTTTTCCGCCGAGGATGCCGAAACCATCGCAAGCCGCACCGTCATGCGTGCGCCCCGCGAAACCTTCCAGTCCGTCGGCTCGATGGTCCAGCAAGCTACGGCCAAGATCGAGCGCGACAGCGTGCAACTGGCTCCCCCTGCCCTGCCCGCGCCATCGGCCGCCGTGGAGCGTTCGCGTCGTCTCGAACAGGAGGCGGCAGGTTTGGCGAAGTCGATGACCATCGACACGCGAGGAACTATGACGACCAAGAAGCGAAAAACCGCCGGCGAGGACCTGGCAAAACAGGTCAGCGAGGCCAAGCAGGCCGCGTTGCTGAAACACACGAAGCAGCAGATCAAGGAAATGCAGCTTTCCTTGTTCGATATTGCGCCGTGGCCGGACACGATGCGAGCGATGCCAAACGACACGGCCCGCTCTGCCCTGTTCACCACGCGCAACAAGAAAATCCCGCGCGAGGCGCTGCAAAACAAGGTCATTTTCCACGTCAACAAGGACGTGAAGATCACCTACACCGGCGTCGAGCTGCGGGCCGACGATGACGAACTGGTGTGGCAGCAGGTGTTGGAGTACGCGAAGCGCACCCCTATCGGCGAGCCGATCACCTTCACGTTCTACGAGCTTTGCCAGGACCTGGGCTGGTCGATCAATGGCCGGTATTACACGAAGGCCGAGGAATGCCTGTCGCGCCTACAGGCGACGGCGATGGGCTTCACGTCCGACCGCGTTGGGCACCTGGAATCGGTGTCGCTGCTGCACCGCTTCCGCGTCCTGGACCGTGGCAAGAAAACGTCCCGTTGCCAGGTCCTGATCGACGAGGAAATCGTCGTGCTGTTTGCTGGCGACCACTACACGAAATTCATATGGGAGAAGTACCGCAAGCTGTCGCCGACGGCCCGACGGATGTTCGACTATTTCAGCTCGCACCGGGAGCCGTACCCGCTCAAGCTGGAAACCTTCCGCCTCATGTGCGGATCGGATTCCACCCGCGTGAAGAAGTGGCGCGAGCAGGTCGGCGAAGCCTGCGAAGAGTTGCGAGGCAGCGGCCTGGTGGAACACGCCTGGGTCAATGATGACCTGGTGCATTGCAAACGCTAGGGCCTTGTGGGGTCAGTTCCGGCTGGGGGTTCAGCAGCCAGCGCTTTACTGGCATTTCAGGAACAAGCGGGCACTGCTCGACGCACTTGCTTCGCTCAGTATCGCTCGGGACGCACGGCGCGCTCTACGAACTGCCGATAAACAGAGGATTAAAATTGACAATTCAATGGCAAGGACTGCCAGCGCTGCCATTTTTGGGGTGAGGCCGTTCGCGGCCGAGGGGCGCAGCCCCTGGGGGGATGGGAGGCCCGCGTTAGCGGGCCGGGAGGGTTCGAGAAGGGGGGGCACCCCCCTTCGGCGTGCGCGGTCACGCGCACAGGGCGCAGCCCTGGTTAAAAACAAGGTTTATAAATATTGGTTTAAAAGCAGGTTAAAAGACAGGTTAGCGGTGGCCGAAAAACGGGCGGAAACCCTTGCAAATGCTGGATTTTCTGCCTGTGGACAGCCCCTCAAATGTCAATAGGTGCGCCCCTCATCTGTCAGCACTCTGCCCCTCAAGTGTCAAGGATCGCGCCCCTCATCTGTCAGTAGTCGCGCCCCTCAAGTGTCAATACCGCAGGGCACTTATCCCCAGGCTTGTCCACATCATCTGTGGGAAACTCGCGTAAAATCAGGCGTTTTCGCCGATTTGCGAGGCTGGCCAGCTCCACGTCGCCGGCCGAAATCGAGCCTGCCCCTCATCTGTCAACGCCGCGCCGGGTGAGTCGGCCCCTCAAGTGTCAACGTCCGCCCCTCATCTGTCAGTGAGGGCCAAGTTTTCCGCGAGGTATCCACAACGCCGGCGGCCGCGGTGTCTCGCACACGGCTTCGACGGCGTTTCTGGCGCGTTTGCAGGGCCATAGACGGCCGCCAGCCCAGCGGCGAGGGCAACCAGCCCGGTGAGCGTCGCAAAGGCGCTCGGTCTTGCCTTGCTCGTCGAGATCTGGGGTCGATCAGCCGGGGATGCATCAGGCCGACAGTCGGAACTTCGGGTCCCCGACCTGTACCATTCGGTGAGCAATGGATAGGGGAGTTGATATCGTCAACGTTCACTTCTAAAGAAATAGCGCCACTCAGCTTCCTCAGCGGCTTTATCCAGCGATTTCCTATTATGTCGGCATAGTTCTCAAGATCGACAGCCTGTCACGGTTAAGCGAGAAATGAATAAGAAGGCTGATAATTCGGATCTCTGCGAGGGAGATGATATTTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGThe sequence indicated in bold above is the portion that encodes the fusion protein:(SEQ ID NO: 6) ATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAAGGAGGTTCTGGTGGATCAGGAGGTCCATCTGGAGGTTCTGGAGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTTTAA

The proteins of the present invention may be expressed from an isolatedDNA sequence encoding the protein. “Recombinant” is defined as a peptideor nucleic acid produced by the processes of genetic engineering. Itshould be noted that it is well-known in the art that, due to theredundancy in the genetic code, individual nucleotides can be readilyexchanged in a codon, and still result in an identical amino acidsequence.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues. A “nucleic acid fragment” is a fraction of agiven nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majorityof organisms is the genetic material while ribonucleic acid (RNA) isinvolved in the transfer of information contained within DNA intoproteins. The term “nucleotide sequence” refers to a polymer of DNA orRNA that can be single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. The terms “nucleic acid,”“nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequenceor segment,” or “polynucleotide” may also be used interchangeably withgene, cDNA, DNA and RNA encoded by a gene.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” DNA molecule or an “isolated” or “purified”polypeptide is a DNA molecule or polypeptide that exists apart from itsnative environment and is therefore not a product of nature. An isolatedDNA molecule or polypeptide may exist in a purified form or may exist ina non-native environment such as, for example, a transgenic host cell orbacteriophage. For example, an “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In oneembodiment, an “isolated” nucleic acid is free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. A protein that is substantiallyfree of cellular material includes preparations of protein orpolypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) ofcontaminating protein. When the protein of the invention, orbiologically active portion thereof, is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of-interestchemicals. Fragments and variants of the disclosed nucleotide sequencesand proteins or partial-length proteins encoded thereby are alsoencompassed by the present invention. By “fragment” or “portion” ismeant a full length or less than full length of the nucleotide sequenceencoding, or the amino acid sequence of, a polypeptide or protein.

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNA,functional RNA, or specific protein, including regulatory sequences.Genes also include nonexpressed DNA segments that, for example, formrecognition sequences for other proteins. Genes can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and mayinclude sequences designed to have desired parameters.

“Naturally occurring” is used to describe an object that can be found innature as distinct from being artificially produced. For example, aprotein or nucleotide sequence present in an organism (including avirus), which can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory, is naturallyoccurring.

The term “chimeric” refers to any gene or DNA that contains 1) DNAsequences, including regulatory and coding sequences that are not foundtogether in nature or 2) sequences encoding parts of proteins notnaturally adjoined, or 3) parts of promoters that are not naturallyadjoined. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orcomprise regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different from that found in nature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, DNA that is either heterologous or homologous to the DNA of aparticular cell to be transformed. Additionally, transgenes may comprisenative genes inserted into a non-native organism, or chimeric genes. Theterm “endogenous gene” refers to a native gene in its natural locationin the genome of an organism. A “foreign” gene refers to a gene notnormally found in the host organism but that is introduced by genetransfer.

A “variant” of a molecule is a sequence that is substantially similar tothe sequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis that encode the native protein, as wellas those that encode a polypeptide having amino acid substitutions.Generally, nucleotide sequence variants of the invention will have atleast 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%,e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to98%, sequence identity to the native (endogenous) nucleotide sequence.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

“Recombinant DNA molecule” is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (3^(rd) edition, 2001).

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” each refer to a sequence that originatesfrom a source foreign to the particular host cell or, if from the samesource, is modified from its original form. Thus, a heterologous gene ina host cell includes a gene that is endogenous to the particular hostcell but has been modified. The terms also include non-naturallyoccurring multiple copies of a naturally occurring DNA sequence. Thus,the terms refer to a DNA segment that is foreign or heterologous to thecell, or homologous to the cell but in a position within the host cellnucleic acid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides.

A “homologous” DNA sequence is a DNA sequence that is naturallyassociated with a host cell into which it is introduced.

“Wild-type” refers to the normal gene, or organism found in naturewithout any known mutation.

Expression Cassettes

In certain embodiments, the present invention provides an expressioncassette comprising the nucleic acid described herein and a promoter.

In certain embodiments, the promoter is a plant promoter.

In certain embodiments, the plant promoter is operable in corn or rice.

In certain embodiments, the plant promoter is operable in seed tissue.

In certain embodiments, the seed tissue is embryo or endosperm tissue.

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest. The expression cassette comprising the nucleotide sequence ofinterest may be chimeric, meaning that at least one of its components isheterologous with respect to at least one of its other components. Theexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.The expression of the nucleotide sequence in the expression cassette maybe under the control of a constitutive promoter or of an induciblepromoter that initiates transcription only when the host cell is exposedto some particular external stimulus. In the case of a multicellularorganism, the promoter can also be specific to a particular tissue ororgan or stage of development.

Such expression cassettes will comprise the transcriptional initiationregion of the invention linked to a nucleotide sequence of interest.Such an expression cassette is provided with a plurality of restrictionsites for insertion of the gene of interest to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences that may be a combination of syntheticand natural sequences. As is noted above, the term “suitable regulatorysequences” is not limited to promoters. However, some suitableregulatory sequences useful in the present invention will include, butare not limited to constitutive promoters, tissue-specific promoters,development-specific promoters, inducible promoters and viral promoters.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency.

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or even becomprised of synthetic DNA segments. A promoter may also contain DNAsequences that are involved in the binding of protein factors thatcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one is affected bythe other. For example, a regulatory DNA sequence is said to be“operably linked to” or “associated with” a DNA sequence that codes foran RNA or a polypeptide if the two sequences are situated such that theregulatory DNA sequence affects expression of the coding DNA sequence(i.e., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation in a cell ofan endogenous gene, transgene, as well as the transcription and stableaccumulation of sense (mRNA) or functional RNA. In the case of antisenseconstructs, expression may refer to the transcription of the antisenseDNA only. Expression may also refer to the production of protein.

“Transcription stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples oftranscription stop fragments are known to the art.

“Translation stop fragment” refers to nucleotide sequences that containone or more regulatory signals, such as one or more termination codonsin all three frames, capable of terminating translation. Insertion of atranslation stop fragment adjacent to or near the initiation codon atthe 5′ end of the coding sequence will result in no translation orimproper translation. Excision of the translation stop fragment bysite-specific recombination will leave a site-specific sequence in thecoding sequence that does not interfere with proper translation usingthe initiation codon.

The terms “cis-acting sequence” and “cis-acting element” refer to DNA orRNA sequences whose functions require them to be on the same molecule.

The terms “trans-acting sequence” and “trans-acting element” refer toDNA or RNA sequences whose function does not require them to be on thesame molecule.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity,” and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a known mathematical algorithm.Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (available on the worldwide web at ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignmentscore falls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, thedefault parameters of the respective programs (e.g., BLASTN fornucleotide sequences, BLASTX for proteins) can be used. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix. See the world-wide-web at ncbi.nlm.nih.gov.Alignment may also be performed manually by visual inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to a specifiedpercentage of residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window,as measured by sequence comparison algorithms or by visual inspection.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%,and at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to areference sequence using one of the alignment programs described usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 70%, at least 80%,90%, at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%,97%, 98% or 99%, sequence identity to the reference sequence over aspecified comparison window. An indication that two peptide sequencesare substantially identical is that one peptide is immunologicallyreactive with antibodies raised against the second peptide. Thus, apeptide is substantially identical to a second peptide, for example,where the two peptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The thermal melting point(T_(m)) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl: T_(m) 81.5° C.+16.6(log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity ofmonovalent cations, % GC is the percentage of guanosine and cytosinenucleotides in the DNA, % form is the percentage of formamide in thehybridization solution, and L is the length of the hybrid in base pairs.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with >90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the T_(m);moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the T_(m); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the T_(m). Using the equation, hybridization and washcompositions, and desired temperature, those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a temperature of less than 45° C. (aqueoussolution) or 32° C. (formamide solution), it is preferred to increasethe SSC concentration so that a higher temperature can be used.Generally, highly stringent hybridization and wash conditions areselected to be about 5EC lower than the T_(m) for the specific sequenceat a defined ionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72ECfor about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65EC for 15 minutes. Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Anexample medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1×SSC at 45EC for 15 minutes. An example low stringencywash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at40EC for 15 minutes. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ionconcentration (or other salts) at pH 7.0 to 8.3, and the temperature istypically at least about 30EC and at least about 60° C. for long probes(e.g., >50 nucleotides). Stringent conditions may also be achieved withthe addition of destabilizing agents such as formamide. In general, asignal to noise ratio of 2× (or higher) than that observed for anunrelated probe in the particular hybridization assay indicatesdetection of a specific hybridization. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the proteins that they encode aresubstantially identical. This occurs, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, thepolypeptides of the invention encompass naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the desired activity. The deletions, insertions, andsubstitutions of the polypeptide sequence encompassed herein are notexpected to produce radical changes in the characteristics of thepolypeptide. However, when it is difficult to predict the exact effectof the substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q). In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence are also “conservatively modified variations.”

In certain embodiments, the nucleic acid sequences are the following:

Fusion protein cds, with signal peptide: (SEQ ID NO: 7)ATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAAGGAGGTTCTGGTGGATCAGGAGGTCCATCTGGAGGTTCTGGAGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTT Fusion protein cds, without signal peptide:(SEQ ID NO: 8)ATGGCTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAAGGAGGTTCTGGTGGATCAGGAGGTCCATCTGGAGGTTCTGGAGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTT 6His-plcC, with signal peptide: (SEQ ID NO: 9)ATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAA 6His-plcC, without signal peptide:(SEQ ID NO: 10)ATGGCTCACCATCACCATCATCACGGATCCGACCCATCCGTGGGAAACAACGTTAAGGAGCTTGTGGCTTACATCTCCACTTCTGGAGAGAAGGACGCTGGAACCGACGATTACATGTACTTCGGTATCAAGACCAAGGATGGAAAGACTCAAGAATGGGAGATGGACAATCCAGGTAACGACTTCATGGCTGGTAGCAAGGATACTTACACTTTCAAGTTGAAAGACGAGAACCTTAAGATCGACGACATCCAGAACATGTGGATTAGGAAACGTAAGTACACCGCCTTCCCAGACGCTTACAAGCCTGAGAACATCAAGGTTATCGCTAACGGAAAGGTGGTTGTTGACAAGGATATCAACGAGTGGATTTCTGGAAACTCCACTTACAACATCAAA 6His-netB, with signal peptide:(SEQ ID NO: 11)ATGGCTAACAAGCACCTCTCATTGTCTCTCTTCCTTGTGCTCCTTGGTCTTTCTGCTTCTCTTGCTTCTGGTCACCATCACCATCATCACGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTT6His-netB, without signal peptide: (SEQ ID NO: 12)ATGGCTCACCATCACCATCATCACGGATCCGAGCTTAACGACATCAACAAGATTGAGCTTAAGAACCTCTCCGGAGAGATCATCAAGGAGAACGGTAAGGAGGCTATCAAGTACACTTCTTCCGACACCGCTTCCCACAAGGGATGGAAGGCCACTCTTTCTGGAACCTTCATCGAAGACCCTCATTCTGACAAGAAGACTGCTTTGCTTAACCTTGAAGGATTCATCCCATCTGACAAACAGATCTTCGGATCTAAGTACTACGGAAAGATGAAGTGGCCTGAGACTTACAGGATCAACGTGAAGAGCGCTGACGTTAACAACAACATCAAGATCGCCAACTCTATTCCGAAGAACACTATCGACAAGAAGGACGTGTCCAATTCTATCGGTTACTCCATCGGAGGTAACATCTCTGTTGAGGGTAAGACTGCTGGTGCTGGAATCAACGCTTCTTACAACGTTCAGAACACTATCTCCTATGAGCAACCTGACTTCAGAACCATTCAGAGGAAGGACGATGCTAACCTTGCATCCTGGGACATCAAATTCGTTGAGACTAAGGACGGATACAACATCGACTCCTACCATGCTATCTATGGCAACCAGCTCTTCATGAAGAGCAGATTGTACAACAATGGTGACAAGAACTTCACCGACGATAGGGACCTCTCCACCTTGATCTCTGGTGGATTCTCTCCAAACATGGCTCTTGCCTTGACCGCTCCTAAGAACGCTAAGGAGTCAGTGATCATCGTTGAATACCAGAGGTTCGACAACGACTATATCCTTAACTGGGAGACTACTCAAGCTAGAGGAACTAACAAGCTTTCTTCAACCTCCGAGTACAACGAGTTTATGTTCAAGATCAACTGGCAGGACCACAAGATCGAATACTATCTT

Vectors

In certain embodiments, the present invention provides a recombinantvector comprising the expression cassette described herein and a vector.

In certain embodiments, the vector is a viral vector.

In certain embodiments, the vector is a bean yellow dwarf virusreplicon.

In certain embodiments, the vector is pBYR2eK2M-6HplcCnetB. (SEQ ID NO:14 and FIG. 6).

A “vector” is defined to include, inter alia, any plasmid, cosmid, phageor binary vector in double or single stranded linear or circular formwhich may or may not be self transmissible or mobilizable, and which cantransform prokaryotic or eukaryotic host either by integration into thecellular genome or exist extrachromosomally (e.g., autonomousreplicating plasmid with an origin of replication).

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

Plant Cells and Animal Feed

In certain embodiments, the present invention provides a plant cellcomprising the antigenic protein described herein, the nucleic aciddescribed herein, the expression cassette described herein or therecombinant vector described herein.

In certain embodiments, the plant is a corn or rice cell.

In certain embodiments, the plant cell further comprises an E. coliheat-labile enterotoxin (LT) and/or a cholera toxin (CT).

In certain embodiments, the present invention provides animal feedcomprising the plant cell described herein.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.

“Transformed,” “transgenic,” and “recombinant” refer to a host cell ororganism into which a heterologous nucleic acid molecule has beenintroduced. The nucleic acid molecule can be stably integrated into thegenome generally known in the art. Known methods of PCR include, but arenot limited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like. Forexample, “transformed,” “transformant,” and “transgenic” cells have beenthrough the transformation process and contain a foreign gene integratedinto their chromosome. The term “untransformed” refers to normal cellsthat have not been through the transformation process.

A “transgenic” organism is an organism having one or more cells thatcontain an expression vector.

Vaccines

In certain embodiments, the present invention provides a vaccinecomprising the antigenic protein described herein, the nucleic aciddescribed herein, the expression cassette described herein, therecombinant vector described herein, the plant cell described herein, orthe animal feed described herein.

The fusion antigen was readily purified using metal affinitychromatography, and used for chicken immunization experiments. The dataindicate that the plant-made fusion protein was immunogenic andprotective. Evidence was observed on western blots that the PlcC-NetBaccumulated in several glycosylated forms. A search of the PlcC-NetBamino acid sequence for consensus Asn-linked glycosylation sites(Asn-X-Ser/Thr) showed one site in the PlcC and four sites in the NetBdomain. Mapping of these sites on the 3-dimensional structures of Plcand NetB showed that they mostly occur in surface loops, and thusprobably would not interfere with correct folding of the proteins orimpair the antigen structure of protective epitopes. In some cases, sucheukaryotic glycosylation was shown to be either neutral in effect orenhance immunogenicity of plant-made antigens. However, it is difficultto predict the effects of glycosylation on the immunogenicity ofPlcC-NetB. The preliminary study showed it is immunogenic in chickens,it is possible that a non-glycosylated protein will be even more potent.

Thus, a new expression vector was constructed that lacks the N-terminalsignal sequence, which resulted in cytosolic accumulation and thusunglycosylated antigen. The glycosylated and unglycosylated antigens areused in further studies to test immunogenicity and protection inchickens. Several mutant forms of NetB have been studied and showedreduced toxicity and may retain protective immunogenicity. Single aminoacid substitutions in the rim loop region that significantly reduce itstoxicity include Y191A, R200A, W257A, W262A S254L, R230Q and W287R. Someof these were shown to retain the ability to generate protective immuneresponses, including W262A and S254L. Thus it is reasonable tocontemplate the use of multiple different mutations in the NetBcomponent of the PlcCNetB fusion protein, in order to maximize itssafety. For production of the fusion protein in seeds of corn or rice,stable transgenic lines must be developed. The expression constructwould use an appropriate promoter that will drive strong expression in aseed tissue, such as embryo or endosperm tissues. Agrobacterium-mediateddelivery of DNA to embryogenic cell cultures enables creation of stablytransformed whole plants that transmit the transgenes to sexual progeny.

One may consider the co-delivery of a mucosal adjuvant to enhanceimmunogenicity of the PlcC-NetB antigens. The E. coli heat-labileenterotoxin (LT) and related cholera toxin (CT) are potent stimulatorsof mucosal immunity. LT and mutants thereof (including LTA S63K and A72Rhave been expressed in transgenic tobacco cells, and were well toleratedand immunogenic in chickens by oral or parenteral delivery. Orallyimmunogenic LT-B was expressed in transgenic corn; and CT-B wasexpressed in transgenic rice. Methods for milling and formulating cornand rice for oral delivery are well developed and convenient.

Methods of Administration

In certain embodiments, the present invention provides a method ofprotecting an avian species from C. perfringens infections comprisingadministering the vaccine described herein.

In certain embodiments, the avian species is chicken, turkey, duck orostrich.

In certain embodiments, the avian is a chicken or turkey.

The present invention also provides a method of protecting poultry byadministering to the poultry an immunologically protective amount of avaccine of the present invention. As used herein, the term“immunologically protective” means that the vaccine is effective ininducing a protective immune response. An immunological response to acomposition or vaccine is the development in the host of a cellularand/or antibody-mediated immune response to the protein or vaccine ofinterest. Usually, such a response consists of the subject producingantibodies, B cell, helper T cells, suppressor T cells, and/or cytotoxicT cells directed specifically to an antigen or antigens included in thecomposition or vaccine of interest.

The fusion can be purified and used to inject birds. Injections can begiven to hens prior to lay, to enhance immunity of chicks during thefirst 2-3 weeks of life by passive transfer of antibodies against thefusion protein. In certain embodiments, a suitable adjuvant is used. Forexample, saponin adjuvant such as Quil A, various oil emulsion adjuvantssuch as water in oil or water in oil in water formulations are used.

The agents of the invention are preferably administered so as to resultin a reduction in at least one symptom associated with a disease. Theamount administered will vary depending on various factors including,but not limited to, the composition chosen, the particular disease, theweight, the physical condition, and the age of the mammal, and whetherprevention or treatment is to be achieved. Such factors can be readilydetermined by the clinician employing animal models or other testsystems, which are well known to the art.

Administration of therapeutic agents may be accomplished through theadministration of the therapeutic agent, such as a fusion protein.Pharmaceutical formulations, dosages and routes of administration forpeptide are generally known.

The present invention envisions treating uveitis in a mammal by theadministration of an agent, e.g., a fusion protein. Administration ofthe therapeutic agents in accordance with the present invention may becontinuous or intermittent, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of the agents of the invention may beessentially continuous over a preselected period of time or may be in aseries of spaced doses. Both local and systemic administration iscontemplated.

One or more suitable unit dosage forms having the therapeutic agent(s)of the invention, which, as discussed below, may optionally beformulated for sustained release (for example using microencapsulation,see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of whichare incorporated by reference herein), can be administered by a varietyof routes including parenteral, including by intravenous andintramuscular routes. The formulations may, where appropriate, beconveniently presented in discrete unit dosage forms and may be preparedby any of the methods well known to pharmacy. Such methods may includethe step of bringing into association the therapeutic agent with liquidcarriers, solid matrices, semi-solid carriers, finely divided solidcarriers or combinations thereof, and then, if necessary, introducing orshaping the product into the desired delivery system.

When the therapeutic agents of the invention are prepared foradministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations include from 0.1 to 99.9% by weight of the formulation. A“pharmaceutically acceptable” is a carrier, diluent, excipient, and/orsalt that is compatible with the other ingredients of the formulation,and not deleterious to the recipient thereof. The active ingredient foradministration may be present as a powder or as granules, as a solution,a suspension or an emulsion.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. The therapeutic agents of theinvention can also be formulated as solutions appropriate for parenteraladministration, for instance by intraocular routes.

The pharmaceutical formulations of the therapeutic agents of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that arewell-known in the art. Specific non-limiting examples of the carriersand/or diluents that are useful in the pharmaceutical formulations ofthe present invention include water and physiologically acceptablebuffered saline solutions such as phosphate buffered saline solutions pH7.0-8.0 saline solutions and water.

As used herein, the term “therapeutic agent” refers to a fusion proteinagent or material containing the fusion protein that has a beneficialeffect on the mammalian recipient. “Treating” as used herein refers topreventing infection of C. perfringens infection.

The present invention also provides a method of protecting poultry byadministering a vaccine that is effective in inducing cellular andhumoral immunity and that contains a biological agent or microbialcomponent that is effective in stimulating a protective cellular andhumoral immune response to C. perfringens.

The purified protein can also be used for in ovo vaccination. Again, asuitable adjuvant may be used to enhance immunogenicity, as discussedabove. The vaccine of the present invention can be administered viaconventional modes of administration or in ovo. Methods of in ovoimmunization are set forth, for example, in U.S. Pat. No. 6,048,535.Vaccination can be performed at any age. For in ovo vaccination,vaccination would be done in the last quarter of embryonal developmentbut may be done at any time during embryonation. The vaccines accordingto the invention can, for example, be administered intramuscularly,subcutaneously, orally, intraocularly, intratracheally, intranasally, inovo, in drinking water, in the form of sprays or by contact spread.Preferably, chickens are given the first vaccine in ovo or at one day ofage. Subsequent vaccinations are done according to need. Breederchickens can be vaccinated before and during the lay cycle (severalinoculations).

In certain embodiments, the vaccine is administered in poultry feed.

In certain embodiments, the vaccine is administered by injection.

In certain embodiments, the vaccine is administered in ovo.

Adjuvants

Vaccines are often formulated and inoculated with various adjuvants. Theadjuvants aid in attaining a more durable and higher level of immunityusing small amounts of antigen or fewer doses than if the immunogen wereadministered alone. The mechanism of adjuvant action is complex, and mayinvolve the stimulation of cytokine production, phagocytosis and otheractivities of the reticuloendothelial system as well as a delayedrelease and degradation of the antigen. Suitable adjuvants include butare not limited to surfactants, e.g., hexadecylamine, octadecylamine,lysolecithin, dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′—N-bis(2-hydroxyethyl-propane di-amine),methoxyhexadecyl-glycerol, and pluronic polyols; polanions, e.g., pyran,dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g.,muramyl dipeptide, aimethylglycine, tuftsin, oil emulsions, alum, andmixtures thereof. Other potential adjuvants include the B peptidesubunits of E. coli heat labile toxin or of the cholera toxin, andmutant forms of complete toxin in which mutations have been introducedinto the A subunit of E. coli heat labile toxin or cholera toxin thatattenuate its toxicity while retaining its adjuvant properties. McGhee,J. R., et al., “On vaccine development,” Sem. Hematol., 30:3-15 (1993).Finally, the immunogenic product may be incorporated into liposomes foruse in a vaccine formulation, or may be conjugated to proteins such askeyhole limpet hemocyanin (KLH) or human serum albumin (HSA) or otherpolymers.

In certain embodiments, a saponin adjuvant such as Quil A, various oilemulsion adjuvants such as water in oil or water in oil in waterformulations are used.

The invention will now be illustrated by the following non-limitingExample.

Example 1

Introduction

Clostridium perfringens (C. perfringens) induced necrotic enteritis (NE)is becoming an economically significant problem for the broilerindustry. The acute form of the disease leads to increased mortality inbroiler flocks, which can account for high losses of up to 1% per day,reaching mortality rates up to 10-40%. In the subclinical form, fibrindeposits and other damage to the intestinal mucosa caused by C.perfringens (FIG. 1) leads to poor productivity (reduced growth, reducedfeed efficiency) without mortality. C. perfringens-infected poultry alsoconstitutes a risk for transmission to humans through the food chain.Historically, C. perfringens outbreaks in the broiler industry wereavoided by the use of growth-promoting antimicrobials in the diet.However, concerns regarding antibiotic resistance led to restrictions onthe use of antibiotics. This, coupled with high-density livingconditions and the reuse of litter materials, has culminated in aresurgence of C. perfringens infections, estimated to cause a globaleconomic loss of over $US2 billion annually.

C. perfringens is a Gram-positive anaerobic spore-forming bacterium. Atleast 17 exotoxins and enzymes responsible for the associated lesionsand disease symptoms have been identified. C. perfringens strains areclassified into five types (A, B, C, D and E), based on their ability toproduce different combinations of four major toxins (α, β, ε and ι). NEand the subclinical form of C. perfringens infection in poultry arecaused by C. perfringens type A strains. For many years, thechromosome-encoded alpha-toxin, a membrane active phospholipase, wasconsidered to be the major toxin associated with NE. Alpha-toxin iscomposed of two domains, which are associated with phospholipase Cactivity (N-domain, 1-250 residues) and membrane recognition (C-domain,251-370 residues), respectively. The C-terminal domain contributes tomaintaining the active form of the toxin and mediates interactions withmembrane phospholipids in a calcium-dependent manner. Individually thesedomains are non-toxic but immunogenic in mice resulting in thegeneration of antibody that reacts with the holotoxin, however, onlyimmune responses against the C-domain provided protection against asubsequent challenge, possibly due to the blocking effects on theinitial membrane-binding event. Therefore, the C-terminal domain of thealpha-toxin has been studied extensively as a vaccine against C.perfringens infection, delivered as a purified protein or by liveattenuated bacteria. Currently the only commercially available vaccinefor necrotic enteritis, Netvax®, is composed of an alpha toxoid derivedfrom a C. perfringens type A strain.

Recent studies have identified a β-like toxin linked to necroticenteritis, designated NetB toxin. It was identified in an Australian C.perfringens type A strain and has been proposed to be the most criticalvirulence factor for the development of NE in broilers. NetB is apore-forming toxin encoded on a large conjugative plasmid (approximately85 kb) within a 42 kilobase (kb) pathogenicity locus (NELoc-1), showingsimilarity to C. perfringens 0-toxin (38% identity). Several studieshave screened for the presence of the netB gene within C. perfringensisolates and found that the presence of netB gene is highly correlatedwith necrotic enteritis strains. NetB is also a protective antigen,which could provide protection against C. perfringens challenge,especially in combination with other immunogenic components. Resultsconsistent with a protective role for immune responses to NetB wereobtained in a study that examined serum antibody levels against C.perfringens alpha-toxin and NetB toxin in commercial birds from fieldoutbreaks of NE. The results showed that the levels of serum antibodiesagainst both alpha-toxin and NetB toxin were significantly higher inapparently healthy chickens compared to birds with clinical signs of NE,suggesting that these antitoxin antibodies may play a role in protectionagainst NE. Their results indicate a correlation between the presence ofantitoxin antibodies in the serum and protective immunity against NE. Inone study, purified α-toxin C-fragment and NetB (W262A) toxoids weremixed (30 μg of each) in Quil A adjuvant and used to subcutaneouslyinject broiler birds 3 times, on days 3, 9 and 15. Birds injected withonly one of the proteins were also included. The immunized birds werepartially protected against a mild gavage challenge, but not against amore severe, in feed challenge. In some studies, hens were infected withNetB toxoid and antibodies against NetB were transferred from immunizedhens to progeny, providing protection against C. perfringens challenge.In another study, immunization with both NetB and α-toxin toxoids usinga live Salmonella delivery vector induced mucosal antibodies againstboth toxins and elicited a protective response. Strains engineered todeliver both toxoids provided significantly better protection thanstrains delivering each toxin alone.

In the current study, the immunogenicity of a novel PlcC-NetB fusionprotein was examined in broiler birds.

Materials and Methods

Growth of C. perfringens. C. perfringens CP4 was cultured in cooked meatmedium (CMM; Difco) and fluid thioglycollate medium (FTG; Difco).

Purification of PlcC, NetB and PlcC-NetB proteins. His-tagged PlcC(Zekarias, B., H. Mo, and R. Curtiss, III. 2008. Recombinant attenuatedSalmonella enterica serovar Typhimurium expressing the carboxy-terminaldomain of alpha toxin from Clostridium perfringens induces protectiveresponses against necrotic enteritis in chickens. Clin Vaccine Immunol15:805-816) and GST-NetB (Jiang, Y., H. Mo, C. Willingham, S. Wang, J.Y. Park, W. Kong, K. L. Roland, and R. Curtiss, 3rd. 2015. ProtectionAgainst Necrotic Enteritis in Broiler Chickens by Regulated DelayedLysis Salmonella Vaccines. Avian diseases 59:475-485) proteins wereprepared from E. coli as described.

A fusion protein PlcC-NetB was designed comprising the followingcomponents. The PlcC component represents aa 248-370 of alpha toxin(GenBank accession AAP15462.1) (SEQ ID NO: 3). The full-length, mature(i.e., after processing) Alpha toxin (GenBank accession AAP15462.1) isthe following (SEQ ID NO: 16):

WDGKIDGTGTHAMIVTQGVSILENDMSKNEPESVRKNLEILKDNMHELQLGSTYPDYDKNAYDLYQDHFWDPDTNNNFSKDNSWYLAYSIPDTGESQIRKFSALARYEWQRGNYKQATFYLGEAMHYFGDIDTPYHPANVTAVDSAGHVKFETFAEERKEQYKINTVGCKTNEDFYADILKNKDFNAWSKEYARGFAKTGKSIYYSHASMSHSWDDWDYAAKVTLANSQKGTAGYIYRFLHDVSEGNDPSVGNNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMAGSKDTYTFKLKDENLKIDDIQNMWIRKRKYTAFPDAYKPENIKVIANGK VVVDKDINEWISGNSTYNIK

The PlcC component, which is aa 248-370 of alpha toxin (GenBankaccession AAP15462.1) (SEQ ID NO: 3) is the following:

DPSVGNNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMAGSKDTYTFKLKDENLKIDDIQNMWIRKRKYTAFPDAYKPENIKVIANGKVVVDKDINEWISGNSTYNIK

The NetB component represents amino acids 31-322 of NetB (GenBankaccession ACN73257.1) (SEQ ID NO: 5). The full-length NetB (GenBankaccession ACN73257.1) is the following (SEQ ID NO: 17):

MKRLKIISITLVLTSVISTSLFSTQTQVFASELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETTQWRGTNKLSSTSEYNEFMFKINWQDHKIEYYL

The NetB component, which is amino acids 31-322 of NetB (GenBankaccession ACN73257.1) (SEQ ID NO: 5) is the following:

SELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETTQWRGTNKLSSTSEYNEFMFKINWQDHKIEYYL

The PlcC component is linked to the NetB component by the peptide linker“GGSGGSGGPSGGSGG” (SEQ ID NO: 4), with NetB on the C-terminal side. A6His tag (HHHHHHH, SEQ ID NO:2) and linker “HHHHHHGS” (SEQ ID NO: 15) isfused to the N-terminus of PlcC (FIG. 2). Because the toxins arenaturally secreted in C. perfringens via a processed N-terminal signalpeptide, we directed the expressed fusion protein to the endoplasmicreticulum (ER) of plant cells, reasoning that correct protein foldingmay be enhanced by the chaperones present in the ER. In order to targetthe fusion protein to the ER of plant cells, the plant signal peptidefrom barley alpha amylase “MANKHLSLSLFLVLLGLSASLASG” (SEQ ID NO:1) isfused to the N-terminus of the 6His tag. Examination of the sequenceusing SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) andselecting “Eukaryotes” indicates that signal peptidase cleavage islikely to occur between positions 24 and 25: ASG-HH.

A plant codon-optimized coding sequence was designed to enable highexpression in a tobacco relative, Nicotiana benthamiana. Codons wereselected that are more frequently used in highly expressed genes oftobacco and Arabidopsis (Geyer, B. C., L. Kannan, I. Cherni, R. R.Woods, H. Soreq, and T. S. Mor. 2010. Transgenic plants as a source forthe bioscavenging enzyme, human butyrylcholinesterase. Plant BiotechnolJ 8:873-886). Sequences were eliminated that could specify RNAprocessing (splicing, polyadenylation) or destabilization. A commercialservice was used for gene synthesis and cloned the fragment via XbaI at5′ and SacI at 3′ into an expression vector based on a bean yellow dwarfvirus replicon, pBYR2eK2M (Diamos, A. G., S. H. Rosenthal, and H. S.Mason. 2016. 5′ and 3′ Untranslated Regions Strongly Enhance Performanceof Geminiviral Replicons in Nicotiana benthamiana Leaves. Front PlantSci 7:200). The resulting construct pBYR2eK2M-6HplcCnetB was verified byDNA sequencing and transformed into the disarmed Agrobacteriumtumefaciens strain EHA105. Transient expression in leaves We performedby Agrobacterium-mediated DNA delivery. Briefly, Agrobacterium cellswere grown overnight in LB media with 50 μg/ml kanamycin and 1 μg/mlrifampicin, and then cells were collected and resuspended in 10 mM2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5 and 10 mM MgSO₄ toOD₆₀₀=0.2. The resulting bacterial suspensions were injected into leavesthrough a small puncture using a syringe without needle (Huang, Z., andH. S. Mason. 2004. Conformational analysis of hepatitis B surfaceantigen fusions in an Agrobacterium-mediated transient expressionsystem. Plant Biotechnol J 2:241-249). The plants were cultured in agrowth room under moderate light at 25° C. for 4 days before leaves wereharvested and weighed.

The leaves were extracted using a blender in 3-fold mass of buffer(phosphate buffered saline pH 7.5 (PBS), 50 mM sodium ascorbate, 1 mMphenylmethylsulfonyl fluoride, 0.1% Triton X-100), and insoluble debriswas removed by centrifugation (10,000×g, 4° C., 15 min). The supernatantwas collected and 1 M phosphoric acid was added while stirring at 4° C.until the pH=4.8, and then 1 M Tris base was added until the supernatantreached pH=7.5. Precipitated material was removed by centrifugation(10,000×g, 4° C., 15 min), and the supernatant containing recombinantPlcC-NetB was subjected to metal affinity chromatography, using Talon®affinity resin (http://www.clontech.com). Bound protein was eluted bywashing the column with 150 mM imidazole, and fractions were assayed byabsorbance at 280 nm. Combined fractions with the highest proteincontent were dialyzed against PBS, pH 7.5, and the A₂₈₀ was measured.Protein concentration was calculated using the theoretical extinctioncoefficient based on the amino acid sequence of the fusion protein.

The ER-targeted construct resulted in high expression and accumulationof soluble PlcC-NetB fusion protein, which was verified by westernblotting using anti-PlcC and anti-NetB antisera (data not shown). Thefusion antigen was readily purified using metal affinity chromatography.

Detection of Antibody Response by Enzyme-Linked Immunosorbent Assay(ELISA)

ELISAs were performed in triplicate as described (Jiang, Y., Q. Kong, K.L. Roland, and R. Curtiss, 3rd. 2014. Membrane vesicles of Clostridiumperfringens type A strains induce innate and adaptive immunity.International journal of medical microbiology: IJMM 304:431-443) todetermine the titer of IgY r against PlcC, NetB and PlcC-NetB in chickensera. Nunc Immunoplate Maxisorb F96 plates (Nalge Nunc, Rochester, N.Y.)were coated overnight at 4° C. with purified proteins at 100 ng/wellsuspended in sodium carbonate-bicarbonate buffer (pH 9.6). The plateswere blocked with Sea Block blocking buffer (Fisher). Sera fromindividual birds were serially diluted in 2-fold steps from an initialdilution of 1:10 in PBS, respectively. After 1 h incubation at 37° C.,wells were washed three times with PBS-0.05% Tween-20. The plates wereincubated with biotinylated IgY (Southern Biotech) antibodies diluted1:10,000 for 1 h at 37° C. Then streptavidin horseradish peroxidaseconjugate (Southern Biotech) was added at a 1:4,000 dilution.2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, KPL, Inc)was then added to develop the reaction. Color development (absorbance)was recorded at 405 nm using a SpectraMax M2 Multi-Mode MicroplateReader (Molecular Devices, LLC). Endpoint titers were expressed as thereciprocal log 2 values as the last sample dilution with an absorbanceof 0.1 OD unit above that for the negative controls.

Chicken Experiments.

All animal experiments were conducted in compliance with the ArizonaState University Institutional Animal Care and Use Committee and theAnimal Welfare Act. Any chickens that had reached a pre-determinedseverity of clinical illness prior to the end of the experiment werehumanely euthanized and necropsied. One-day-old Cornish×Rock broilerchickens were purchased from Murray McMurray Hatchery (Webster City,Iowa) and typically arrived at our facility at 2 days of age. Birds wererandomly sorted and placed in pens with pine shavings on the floor. Foodand water was supplied ad libitum.

Experiment 1. One week old broiler birds were vaccinated subcutaneouslythree times at weekly intervals with 50 μg of purified PlcC-NetB fusionprotein plus 50 μg of Quil A as adjuvant. The first immunization was at1 week of age. Control birds mock-vaccinated with Quil A only. Thevolume was 100 μl for all inoculations.

Experiment 2. Broiler birds were vaccinated subcutaneously three timesat weekly intervals with 100 μg of purified PlcC-NetB fusion proteinplus 50 μg of Quil A as adjuvant. Control birds mock-vaccinated withQuil A only. The volume was 100 μl for 1^(st) and 2^(nd) inoculationsand 200 μl for 3^(rd) inoculation due to the lower concentration ofprotein in that preparation.

In Experiment 1, serum was taken one week after the final immunizationand assayed for IgY antibodies against PlcC, NetB and PlcC-NetB fusionprotein.

Challenge procedure. The in-feed challenge performed as describedpreviously (Jiang, Y., H. Mo, C. Willingham, S. Wang, J. Y. Park, W.Kong, K. L. Roland, and R. Curtiss, 3rd. 2015. Protection AgainstNecrotic Enteritis in Broiler Chickens by Regulated Delayed LysisSalmonella Vaccines. Avian diseases 59:475-485; Shojadoost, B., A. R.Vince, and J. F. Prescott. 2012. The successful experimental inductionof necrotic enteritis in chickens by Clostridium perfringens: a criticalreview. Vet Res 43:74). Three weeks after the first immunization, birdswere challenged in-feed for 5 days with C. perfringens CP4, a virulentstrain isolated from a necrotic enteritis outbreak. The day after thefinal challenge birds were euthanized and necropsies performed. Atnecropsy, intestinal tracts were examined and scored for lesions typicalof necrotic enteritis. The person performing the scoring was blinded tothe treatment regimen each bird received. Intestinal lesions are scoredas follows: 0=no gross lesions; 1=thin or friable wall or diffusesuperficial but removable fibrin; 2=focal necrosis or ulceration, ornon-removable fibrin deposit, 1 to 5 foci; 3=focal necrosis orulceration, or non-removable fibrin deposit, 6 to 15 foci; 4=focalnecrosis or ulceration, or non-removable fibrin deposit, 16 or morefoci; 5=patches of necrosis 2 to 3 cm long; 6=diffuse necrosis typicalof field cases.

Results

PlcC-NetB protein production in Nicotiana benthamiana. A codon-optimizedgene was designed (FIG. 2) for expression of PlcC-NetB in Nicotianabenthamiana, and it was cloned in an expression vector based on a beanyellow dwarf virus replicon (Diamos, A. G., S. H. Rosenthal, and H. S.Mason. 2016. 5′ and 3′ Untranslated Regions Strongly Enhance Performanceof Geminiviral Replicons in Nicotiana benthamiana Leaves. Front PlantSci 7:200). The system uses transient expression in leaves, withamplified DNA and greatly enhanced protein expression only four daysafter Agrobacterium-mediated DNA delivery. Because the toxins arenaturally secreted in C. perfringens via a processed N-terminal signalpeptide, the expressed fusion protein was directed to the ER of plantcells using a barley alpha-amylase signal peptide, reasoning thatcorrect protein folding may be enhanced by the chaperones present in theER. The construct resulted in high expression and accumulation ofsoluble PlcC-NetB fusion protein, which was readily purified using metalaffinity chromatography, and used for a preliminary chicken immunizationexperiment (see below). The data indicate that the plant-made fusionprotein was immunogenic.

Evidence was observed on western blots that the PlcC-NetB accumulated inseveral glycosylated forms (FIG. 3). A search of the PlcC-NetB aminoacid sequence for consensus Asn-linked glycosylation sites(Asn-X-Ser/Thr) showed one site in the PlcC and four sites in the NetBdomain. Mapping of these sites on the 3-dimensional structures of Plcand NetB showed that they mostly occur in surface loops, and thusprobably would not interfere with correct folding of the proteins orimpair the antigen structure of protective epitopes. In some cases, sucheukaryotic glycosylation was shown to be either neutral in effect orenhance immunogenicity of plant-made antigens (Boes, A., H. Spiegel, G.Edgue, S. Kapelski, M. Scheuermayer, R. Fendel, E. Remarque, F. Altmann,D. Maresch, A. Reimann, G. Pradel, S. Schillberg, and R. Fischer. 2015.Detailed functional characterization of glycosylated and nonglycosylatedvariants of malaria vaccine candidate PfAMA1 produced in Nicotianabenthamiana and analysis of growth inhibitory responses in rabbits.Plant Biotechnol J 13:222-234.; Joensuu, J. J., M. Kotiaho, T. H. Teeri,L. Valmu, A. M. Nuutila, K. M. Oksman-Caldentey, and V. Niklander-Teeri.2006. Glycosylated F4 (K88) fimbrial adhesin FaeG expressed in barleyendosperm induces ETEC-neutralizing antibodies in mice. Transgenic Res15:359-373; Yuki, Y., M. Mejima, S. Kurokawa, T. Hiroiwa, Y. Takahashi,D. Tokuhara, T. Nochi, Y. Katakai, M. Kuroda, N. Takeyama, K. Kashima,M. Abe, Y. Chen, U. Nakanishi, T. Masumura, Y. Takeuchi, H. Kozuka-Hata,H. Shibata, M. Oyama, K. Tanaka, and H. Kiyono. 2013. Induction oftoxin-specific neutralizing immunity by molecularly uniform rice-basedoral cholera toxin B subunit vaccine without plant-associated sugarmodification. Plant Biotechnol J 11:799-808). However, it is difficultto predict the effects of glycosylation on the immunogenicity ofPlcC-NetB. Although the preliminary study showed it is immunogenic inchickens, it is possible that a non-glycosylated protein will be evenmore potent. Thus, a new expression vector was constructed that lacksthe N-terminal signal sequence, which resulted in cytosolic accumulationand thus unglycosylated antigen. The glycosylated and unglycosylatedantigens are used in further studies to test immunogenicity andprotection in chickens.

Serum antibody responses to the PlcC-NetB fusion protein. Serum IgYresponses against the PlcC-NetB protein were significantly higher inimmunized birds compared to non-vaccinated controls (FIG. 4A),indicating that PlcC-NetB is highly immunogenic. However, the protein isglycosylated (FIG. 3) and some of the reacting antibodies could beagainst the carbohydrate moieties, which are not present in thecorresponding proteins produced by C. perfringens. To examine theresponses against the proteinaceous epitopes, PlcC and NetB proteinspurified from E. coli were used as the coating antigen. Although thetiters were somewhat lower, they remained significantly higher thantiters from control animals, indicating that protein epitopes in thePlcC (FIG. 4B) and NetB (FIG. 4C) were being recognized.

Protection Against C. perfringens Challenge.

The results from both challenge experiments are summarized below inTable 1 and graphically in FIG. 4. The challenge in Experiment 1 wasmilder than in Experiment 2, based on the fact that in Experiment 1,none of the birds in the control group received a lesion score of 5.This was due to the fact that different subclones of CP4 were used ineach experiment. Interestingly, the vaccinated birds in Experiment 2 hadoverall healthier intestinal tracts than in Experiment 1. In Experiment1 in which the birds received three doses of 50 μg of PlcC-NetB, afterchallenge, the intestines of most of the vaccinated birds displayedfriability, even in the absence of fibrin. In Experiment 2, where thebirds received three doses of 100 μg of PlcC-NetB, there was littlefriability and only scattered, removable fibrin. This is remarkableconsidering that the challenge was stronger in Experiment 2. Theseresults demonstrate that the PlcC-NetB protein is highly immunogenic andprotective against an in-feed challenge with a highly virulent C.perfringens strain.

TABLE 1 Lesion scores in immunized and non-immunized birds Lesion ScoreAverage Group 0 1 2 3 4 5 6 Lesion Score Exp. 1 PlcC-NetB 3 2 2 0 0 0 0 0.9* Mock 0 0 3 4 3 0 0 3.0 Exp. 2 PlcC-NetB 4 9 0 0 0 0 0  0.7* Mock 00 1 3 4 2 0 3.7 *Different from controls, P = 0.0004 by Mann-Whitneytest **Different from controls, P < 0.001 by Mann-Whitney testExperiment 1: n = 7, PlcC-NetB group; n = 10, mock vaccinated groupExperiment 2: n = 13, PlcC-NetB group; n = 10, mock vaccinated group

Although the foregoing specification and examples fully disclose andenable the present invention, they are not intended to limit the scopeof the invention, which is defined by the claims appended hereto.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. A nucleic acid encoding an antigenic proteincomprising a PlcC protein unit that is operably linked to a peptidelinker that is operably linked to a NetB protein unit, wherein the PlcCprotein unit, the peptide linker and the NetB protein unit each have anN-terminus and a C-terminus, wherein the C-terminus of the PlcC proteinunit is linked to the N-terminus of the peptide linker, wherein theC-terminus of the peptide linker is operably linked to the N-terminus ofthe NetB protein unit, and wherein the peptide linker has at least 95%sequence identity to SEQ ID NO:
 4. 2. The antigenic protein of claim 1,wherein the PlcC protein unit has at least 95% sequence identity to SEQID NO:
 3. 3. The antigenic protein of claim 1, wherein the NetB proteinunit has at least 95% sequence identity to SEQ ID NO:
 5. 4. Theantigenic protein of claim 3, wherein the NetB protein unit has one ormore amino acid substitutions at Y191A, R200A, W257A and W262A, S254L,R230Q or W287R of SEQ ID NO:
 5. 5. The antigenic protein of claim 1,further comprising a 6Hist tag (SEQ ID NO: 2) having an N-terminus and aC-terminus, wherein the C-terminus of the 6Hist tag is operably linkedto the N-terminus of the PlcC protein unit.
 6. The antigenic protein ofclaim 5, further comprising a plant signal peptide having an N-terminusand a C-terminus, wherein the C-terminus of the plant signal peptide isoperably linked to the N-terminus of the 6Hist tag, wherein the plantsignal peptide has at least 95% sequence identity to SEQ ID NO: 1
 7. Thenucleic acid of claim 1, wherein the nucleic acid has at least 95%sequence identity to SEQ ID NO:
 6. 8. An expression cassette comprisingthe nucleic acid of claim 1 and a promoter, wherein the promoter is aplant promoter.
 9. The expression cassette of claim 8, wherein the plantpromoter is operable in corn or rice.
 10. The expression cassette ofclaim 8, wherein the plant promoter is operable in seed tissue.
 11. Arecombinant vector comprising the expression cassette of claim 8 and avector.
 12. The recombinant vector of claim 11, wherein the vector is abean yellow dwarf virus replicon.
 13. A plant cell comprising theantigenic protein of claim
 1. 14. The plant cell of claim 13, furthercomprising an E. coli heat-labile enterotoxin (LT) and/or a choleratoxin (CT).
 15. Animal feed comprising the plant cell of claim 13.